Abstract This paper describes the effects of nanosecond pulsed electric fields (nsPEFs) on the structure and enzyme activity of three kinds of proteins. Intense (up to 300 kV/cm), 5-ns-long electrical pulses were applied to solutions of lysozyme (14 kDa, monomer), albumin (67 kDa, monomer), and urease (480 kDa, hexamer). We analyzed the tertiary and quaternary structures of these proteins as well as their enzyme activity. The results indicated the deformation of both the quaternary and tertiary structures of urease upon exposure to an electric field of 250 kV/cm or more, whereas no structural changes were observed in lysozyme or albumin, even at 300 kV/cm. The enzyme activity of urease also decreased at field strengths of 250 kV/cm or more. Our experiments demonstrated that intense nsPEFs physically affect the conformation and function of some kinds of proteins. Such intense electric fields often occur on cell membranes when these are exposed to a moderate pulsed electric field.